Sunday, 14 September 2014

Two new virology labs: Pirbright's Plowright and the CVR's Sir Michael Stoker

Walter Plowright and Sir Michael Stoker: two names inextricably linked with virology. Their names have now been given to two new buildings that have now been more or less completed. 

Tomorrow our lab moves to the new Sir Michael Stoker building in Glasgow. Ultimately, the building of this new lab will see the entirety of the Centre for Virus Research based on one site, which will make life much easier for people like me who, until now, use pieces of kit from both sites. 
It's gold. Very gold. But it's what's inside that counts. Expansive labs, a big ACDP 3 suite (including insectory), a next generation sequencing suite, a series of rooms for various forms of electron microscopy, and a big server to excite the bioinformaticians, it's an impressive building. Everyone should be excited about working there.

Photo: The final countdown...the contractors are busy with the final preparations before handing over the new Sir Michael Stoker building.
A Golden Temple of virology: the Sir Michael Stoker Building, linked directly with the Sir Henry Wellcome Building
Sir Michael Stoker was chair of virology at the Glasgow MRC virology unit. It is his work with cells for which he is most famed. Originally as a way of researching viruses causing cancer, his work yielded the hamster-derived BHK cell line. It is hard to quantify the impact that this had. Until that point, foot and mouth disease virus (FMDV) vaccines were being produced by growing the viruses in sheets of epithelium that had been peeled off of cows tongues. The BHK cell line, in addition to being generally permissive for many viruses, now enabled the mass production of FMDV vaccines.

Sir Michael Stoker (source)
Regardless of whether it was the Institute for Animal Health (now The Pirbright Institute) or Merial that was responsible for the leak of FMDV, it was already apparent by 2007 that the facilities at The Pirbright Institute were in need of an update. Plans for a redevelopment were already under way, but the outbreak simply served to focus this objective, as well as instigating a reassessment of where the institute's future lay (purely virology - at the time bacteriology and parasitology we firmly established at the sister site at Compton). Last week I went to the centenary event of the Institute, and this included a tour of the newly complete Plowright building. 

In contrast to the relative compactness of the Sir Michael Stoker building, The Plowright building is vast. And it needs to be. Unlike the Stoker building, much of the Plowright building operates at negative pressure in order to allow work on the highest category of animal pathogen, including viruses such as FMDV and African Swine Fever virus. To allow this, virtually a floor of the building is taken up by the fans, filters and pipes of the air handling system. Ultimately, this results in labs that aren't actually that big, and space will be an issue, but walking around it's mighty impressive. For example, the lab I did my PhD in was made of bricks and mortar; this lab is made of a specific concrete that is designed to have fewer air bubbles. 
The Plowright building is also different to most other containment labs of this level. Others, such as AAHL at Geelong, operate as a 'box within a box'; in the case of the Plowright it's been designed so that the outer surface is sufficiently robust that it acts as the outer shell, meaning that the working conditions inside are much more pleasant.

Plowright's vaccine combated rinderpest, which had ravaged herds in Africa.
Walter Plowright (RODNEY WHITE / Associated Press)
In June 2011 the FAO declared the world free of rinderpest. It was only the second time, after smallpox, that such a disease had been wiped from the earth, at least outside the bounds of a laboratory freezer. Fundamental to the eradication of rinderpest, as for smallpox and soon hopefully polio, a vaccine was imperative. And it was Plowright's vaccine that was used for the eradication. It was a great vaccine, being both cheap to produce and resulting in lifelong immunity. The vaccine was derived 'simply' by passaging the virus multiple times in cell culture; something which wasn't quite so 'simple' during the 1950s and 1960s. It was then a huge achievement, and one without which the world may still be plagued by rinderpest.

In the end though, you can have the best building ever, but this it's useless unless it's full of good people doing good science and, with the infrastructure sorted, that's now the focus for everyone.

Sunday, 17 August 2014

Where did Ebola come from? Rooting the un-rootable
As with other emerging viruses, the manner in which Ebolavirus outbreaks appear seemingly from nowhere merely adds to their terror. What is especially intriguing about the current outbreak in West Africa is that it's the wrong virus in the wrong place: as a strain of Zaire Ebolavirus, we would expect to see this virus in central Africa. The modern way to answer the 'where did it come from' question is to compare the genomic sequences of different viruses, and this was one of the first things done for the Guinea isolates. 

The problem is where to root the tree. There have been two schools of thought on why outbreaks of Ebola in Africa happen where they do. Firstly, it's simply a case that Ebola is widely distributed and it is a unfortunate event, such as butchering wild animals, that results in spillover from animals into humans. An alternative, is that Ebola is spreading in a wave across Africa, resulting in outbreaks as it progresses, breaking through at points of weakness. For the latter hypothesis we would expect all of the viruses to relate in a straightforward and logical manner.

Crest of a wave: a figure depicting the hypothesised spread of Ebolavirus in a wave-like fashion. Source
Drawing a straightforward tree with the sequences from the current outbreak along with the available genome sequences of all Ebolaviruses confirms that the virus causing the outbreak in western Africa is a divergent strain of Ebola Zaire, suggesting it arose in central Africa. However, it's out on its own relative to the main Ebola Zaire clade.

The Ebolavirus tree: assembling a tree using all species of Ebolavirus results in the Guinea sequences separated from the remainder of the Ebola Zaire clade. Source

The key, it seems, appears to be to remove the intergenic sequences that separate the coding sequences in the virus. When you do this, and concatenate the coding sequences, the sequences from the Guinea outbreak sit in the middle of the Ebola Zaire clade. If you do the same with the intergenic sequences alone you get a tree with similar topology. The issue the authors encounter is that there is no good place from which to root the tree - the other Ebolavirus species are essentially too distant. Combined with the fact that the viruses are always evolving, this all makes it difficult to see what's really happening.

One alternative is to use time, in combination with the estimated rate of evolution, as a way in which to organise the sequences. Using this molecular clock approach results in an intriguing figure whereby the Guinea sequences do indeed come out up top, where we would expect them to be.

Using a molecular clock to arrange Ebola Zaire sequences places Guinea 2014 furthest from the first isolation in 1976. Source
One thing that this approach also allows is an estimation as to when the current virus diverged from the central African sequences. Dudas and Rambaut estimate 2002, whereas a separate analysis by Calvignac-Spencer et al, also using molecular clocks to root the Ebola Zaire clade, suggest either 1999 or 2001 (depending upon the assumptions made). 

The current outbreak is unprecedented in scale, and it's hard to believe that, when the outbreak is over, there will not be a large study using many sequences. This in turn should give some more clues about how the Ebola wave crashed upon Western Africa.

Dudas G, & Rambaut A (2014). Phylogenetic Analysis of Guinea 2014 EBOV Ebolavirus Outbreak. PLoS currents, 6 PMID: 24860690

Monday, 30 June 2014

A not so curious coincidence: another tick, another phlebovirus.
In recent years there seems to have been a resurgence in Bunyaviruses. There are always incidences of certain members, for example there are thousands of cases of hantavirus each year, as well as the sporadic cases of Crimean-Congo hemorrhagic fever virus. On the other hand there are the new emergents. In Europe, the most dramatic and devastating (at least in animals) has been Schmallenberg virus, which will no doubt now be familiar to more or less all cattle and sheep farmers. 

As far as 'human' viruses are concerned, there have been a couple of highly related viruses: one in the Far East, and one the US. Severe fever with thrombocytopoenia syndrome virus (SFTSV) emerged in China in 2009, and was isolated from one of a series of patients with hemorrhagic fever. The virus from the US is Heartland virus (HV), and was isolated in 2009 from two cases in Missouri. The last few weeks have seen the second death in the US from Heatland virus, this time in Oaklahoma

SFTSV and HV are both members of the phlebovirus genus of the Bunyaviridae. Rift valley fever virus (RVFV), currently one of the great fears for Europe, is also a member of this genus and is spread by mosquitoes. However, unlike RVFV, both SFTSV and HV are associated with ixoid and lone star ticks respectively.

Distribution of the Lone Star tick in the US.

Now a paper has been published describing another tick-borne phlebovirus, named Hunter Island Group virus (HIGV, after the location from which the ticks were sampled), this time from ticks associated with shy albatrosses on an island near Tasmania. Initially an aetiological agent couldn't be found; likely viruses such as Newcastle's disease virus and avian influenza were eliminated as likely causes. Although electron micrographs indicated a virus with morphology suggestive of a bunyavirus, antibody and PCR assays still failed to find a virus. 

Electron microscopic examination results of a newly isolated virus, tentatively named Hunter Island Group virus, isolated from ticks collected from shy albatrosses, Tasmania, Australia. A) Negative-contrast staining of virions. B) Thin section of infected Vero cells showing the presence of viral particles. Original magnification ×100,000; scale bars represent 100 nm,
Electron micrograph images of negatively stained virions (A) and thin sections of vero cell cultures (B). source

Several years later they performed deep sequencing on infected cultures. Although the nucleotide sequences failed to match with anything close, blast searches using the protein sequence revealed similarity with SFTSV. With the foundations of the sequence now known the remaining sequence was filled in, allowing an analysis using the whole genome, as well as the design of molecular assays for the specific detection of HIGV. Perhaps unsurprisingly considering the results from the deep sequencing, the viruses fit into the bunyavirus clade next to SFTSV and Heartland virus. 

Phylogenetic trees of recently isolated bunyaviruses based on amino acid sequences of the polymerase protein (A) encoded by the large segment, the membrane glycoprotein polyprotein (B) encoded by the medium segment, and the nucleocapsid protein (C) and the nonstructural protein (D) encoded by the small segment of selected bunyaviruses. Maximum-likelihood trees were constructed by using MEGA5 ( with bootstrapping at 1,000 replicates. GenBank accession numbers are with
HIGV: phylogenetic trees (amino acid sequence) of the viral polymerase (A), glycoprotein (B), nucleocapsid (C) and NSs (D). source

As the ticks were associated with healthy as well as diseased birds, it was suspected that HIGV wasn't the cause of the outbreak among the shy albatrosses, something which was confirmed when the birds were tested for antibodies against, or the genome of, HIGV. 

Richard Elliott, a well known figure in the bunyavirus  world, says the discovery of this virus wasn't a surprise, and in a way was predicted. The simple truth is that if the ticks are there, then the viruses are there.  

Discussing tick viruses with anyone always seems to leave one unanswered question; what is it that's so repulsive about ticks? 

Wang J, Selleck P, Yu M, Ha W, Rootes C, Gales R, Wise T, Crameri S, Chen H, Broz I, Hyatt A, Woods R, Meehan B, McCullough S, & Wang LF (2014). Novel phlebovirus with zoonotic potential isolated from ticks, australia. Emerging infectious diseases, 20 (6), 1040-3 PMID: 24856477

Sunday, 1 June 2014

Through the looking glass: Guinea, Ebola and life before germ theory

Last week I was at a conference dedicated to viral zoonoses where the opening talk of the meeting was given by Pierre Rollin. Rollin has for a long time been based in the Viral Special Pathogens branch at the CDC in Atlanta, USA. He’s an old hand in outbreaks of savage viruses; a veteran of many Ebola outbreaks, as well as Nipah etc. All in all, he’s someone who can speak from experience. The talk itself ranged across a variety of aspects regarding Ebola virus but concentrated, unsurprisingly, upon the control of outbreaks. Part of the talk was also dedicated to describing the situation in Guinea, from where he’d just returned. 

Health specialists work in an isolation ward for patients in southern Guinea. Photo: 1 April 2014
Ebola in Guinea: hot work, but preventing contact means preventing infection
It’s always more interesting to hear the voice of experience. Books such as The Hot Zone and films such as Outbreak are there to make money and must therefore offer drama; in the case of Ebola endless people with liquefied livers bleeding from every cut and orifice. Rollin pointed out that this is rubbish. Sure, a few do bleed, but only a “minority”; more usually it’s shock and multi organ failure. Gruesome, but not quite as graphic.
One particular point that resonated in the talk was how, in principle, outbreaks of Ebola were easy to control. Find the village, find and isolate those infected and suspected of being infected, plus educate the local population. As remarkably infectious as Ebola is, you need contact with the patient for transmission to occur – sleeping in the same room as an Ebola may be fine (if not recommended), but contact, such as sharing a bed, is a very bad idea. However, if reality were that simple Guinea wouldn't be staring at the prospect of 200 deaths. Speaking to Rollin the next day he confirmed that it was the small details and logistics that caused the most problems. Arriving at a hospital, for example, and finding that the water system is broken. No cleaning. No disinfection. Suddenly something as simple as making up bleach is a challenge. This, though, is a practicality that can be sorted. More difficult is the human aspect: educating the local population. 

 Protective Role for Antibodies in Ebola Vaccine Study Discovered
Education: a poster describing what to (and not to) do regarding Ebola for local population. Image:Medindia.
A recurring theme in Ebola outbreaks is that a lot of cases arise from two population types: healthcare workers, as a result of contact with patients, and, secondly, those involved in traditional practices, such as local ritual burial.
Ritual beliefs still hold fast in many parts of rural Africa. Some locals apparently believe that white man is bringing the disease and is deliberately infecting them. Apparently there are pockets of people in the forests of Guinea who have been hiding bodies from the doctors. In other cases there is a belief that it is spirits and spells that cause the pestilence. In general there’s little understanding of the concept of infectious disease: how many people with even limited knowledge that Ebola is caused by a virus would hug and kiss corpses who had died as a result of the infection? As strong as familial love may be, I think the rational decision may prevail. As a result of all this, there are visits to the likes of witchdoctors and herbal healers that, in turn, become infected and represent a hub of infection for many others.

File:Cholera art.jpg
Miasmas responsible for a cholera epidemic.
To many in a more developed world, this may all sound shockingly primitive. In reality however, this is simply knowledge and education. It was only around the middle of the 19th century that the likes of Louis Pasteur and subsequently Robert Koch really established the germ theory of disease. Prior to this theories of miasmas and mysterious airs still abounded around the world; the ‘mala-aria’ (bad air) derivation of malaria perhaps being one of the most well known. In one sense then, what we are seeing in Guinea is simply life before germ theory. That’s not to say they’re in any way intellectually inferior, more that it is a demonstration that knowledge gathered in more developed nations simply hasn't found its way to other nations. Remoteness inevitably is a factor. But if ever there were an argument for open access journals freely available worldwide....

Sunday, 18 May 2014

Mx2: why some species, and not others?
To answer a question recently posed at a conference, some viruses are clearly good. In general though, becoming infected is something that is to be avoided. In an attempt to prevent infection, cells are armed with the interferon system. Interferon is a molecule that is made and secreted by a cell when it discovers that it's become infected. In turn other cells, yet to become infected, are alerted that an infection is present. This early warning results in the cell producing diverse collection of antiviral molecules, termed interferon stimulated genes (ISGs), each with their own mode of action. There are hundreds of genes upregulated as a result of IFN signalling, but finding out which ISG does what and how is more difficult. One way in which this has been looked at is by expressing ISGs individually and then looking to see whether it has a protective effect against a virus of choice. This approach has led to the identification of various genes such as tetherin, which has since been shown to restrict the progress of multiple viruses from several virus families. Altogether, the action of ISGs, and the various countermeasures of viruses, is a fascinating battle.

At any one point there always seems to be a fashionable ISG. Recently it's been myxovirus 2 (Mx2). the fact that Mx proteins are antiviral is not new. However, two papers came out last year revealing that Mx2 specifically is an ISG that is able to antagonise HIV-1 (so often the virus leading the way). Recently, an Mx2 paper by Busnadiego et al has been published detailing the host and virus specific factors that determine its function. 
The most revealing aspect of this paper is the variation that exists between the Mx2 of different species to restrict HIV-1, in essence a 'human' virus. The authors confirmed that human Mx2 blocked HIV, then tested the Mx2 orthologues from African green monkey (which did restrict), Macaque (which did restrict), ovine (which did NOT restrict) and canine (which also did NOT restrict HIV).

Restriction of HIV-1 by Mx2 from various species: A) evidence that the Mx2 genes are expressed at similar levels. B) reduced titres in the presence of the Mx2 (grey bars) from various species. 

In addition to variation at the host level, the authors also tried a variety of other retroviruses. Whilst other retroviruses weren't restricted by the Mx2 of any species, there was evidence that the Mason pfizer monkey  virus (a betaretrovirus, rather than lentivirus) was sensitive, with varying degrees, to the Mx2 of macaques, African green monkeys and human Mx2. HIV-1 group O, as opposed to group M (which the previous studies had used) was also restricted by human Mx2. However, this group O virus was only partially restricted by African green monkey Mx2, clearly showing species variation. The paper goes on to describe what lies behind these difference in the action of different Mx2s against different viruses.

One way to see which part of the virus is involved in the restriction process is to passage the virus in the presence of the restriction factor. When the authors did this, and sequenced the virus, they found three substitutions in the C-terminal domain of the capsid region within the viral gag gene. By making viruses with each of these mutations in isolation, as well as screening a library of capsid mutants, it could be shown that substituting individual amino acids in the virus sequence is sufficient to direct the specificity of action of different Mx2 proteins. Clearly though, a library can only reveal so much; there are no doubt other positions within, and perhaps outside of, capsid that influence restriction.

Which parts of the Mx2 protein from different species was responsible for the variation that exists in the extent of restriction? One aspect that varied between the Mx2 of different species was its localisation within the cell. Whereas the Mx2 variants that restricted localised to the nuclear pores, those that didn't restrict, i.e. canine and ovine, failed to consistently show such a precise localisation. 

Localisation to the nuclear pore of Mx2: Human Mx2 localises with nuclear pores (Nup98, red) whereas canine, and to an even greater extent ovine, Mx2 orthologs show a weaker association.

The result lack of restriction therefore makes sense, particularly when they show that several amino acids in primate Mx2 sequences, which have been shown to be important for function, (and likely nuclear localisation) are divergent in the ovine and canine versions. Making human-canine chimeric (i.e. part human, part canine) versions of Mx2 narrowed the variability down to the N-terminal end; an Mx2 with a N-terminus from human, but C-terminus from dogs essentially functioned like human Mx2 (capable of restriction, localised to nuclear pores), whereas the opposite, with a canine N-terminus, was unable to restrict, even though the other half was human. Equivalent chimeras between human and African green monkey Mx2, which have differing abilities to restrict a particular clone of HIV-1 revealed a similar story. Ultimately, all of the experiments narrowed down the capability of restriction (at least for these species), to just 3 amino acids in an 8 amino acid region within the N-terminus.

Variation in the Mx2 sequence: Top) diagram of the Mx2 layout with arrows indicating amino acids under positive selection. Bottom) dN/dS and Bayes factor values across the length of Mx2. Low values are indicative of purifying selection. Variation is clearly apparent towards the N-terminus. 
The authors managed to narrow it down further by doing some sequence analyses. Using the primate sequences, they found evidence of positive selection, suggesting some form of arms race between virus and restriction factor. Of 10 amino acids identified as being under positive selection, two occurred in the 8 amino acid region identified using chimeras. By making mutants it was then possible to show that substituting amino acid 37 was sufficient to swap the specificity of human vs. African green monkey Mx2 proteins. This is, though, one example against one mutant virus. There are inevitably many more points of variation dictating species specificity.

Modern technology and methods increasingly allows us to pick apart systems in ever finer detail, and as far as viruses are concerned they don't get more significant than the interferon system; there's a reason viruses go to great lengths to prevent, avoid or overcome it. Picking apart the arsenal of ISGs, the front-line effectors of the system, may reveal more and more the variation that exists between species and, in turn, help to answer a fascinating question regarding virus host range: 'why some species, and not others?'.

Busnadiego, I., Kane, M., Rihn, S., Preugschas, H., Hughes, J., Blanco-Melo, D., Strouvelle, V., Zang, T., Willett, B., Boutell, C., Bieniasz, P., & Wilson, S. (2014). Host and Viral Determinants of Mx2 Antiretroviral Activity Journal of Virology DOI: 10.1128/JVI.00214-14

Monday, 10 March 2014

Rift Valley fever virus genome organisation: it's like it for a reason
Following recent Bluetongue virus and Schmallenberg virus (SBV) incursions into Central and Northern Europe, Rift Valley Fever virus (RVFV) is now perceived as one of the greatest threats to Europe, crossing over the Mediterranean from Africa where it remains endemic. With good reason. Both Culex  and Aedes species of mosquito are capable of transmitting the virus - West Nile virus infections in Italy have previously shown that there are conditions in Europe suitable for arbovirus transmission by such species of mosquito.
Like SBV, which has spread across Europe in the last 2-3 years, RVFV is a member of the Bunyaviridae family, (albeit in the genus Phlebovirus as opposed to Orthobunyavirus). If it were to encroach into Europe, the headline fact is that it's a zoonosis, with the potential to cause fatal disease in humans. Of equal, if not more, importance is the that fact that it can cause deaths and abortions in ruminants.

Dead cattle as a result of RVFV infection (

The RVFV genome comprises three segments of (-)ssRNA. Although the small (S) segment encodes both the nucleocapsid (N) protein and the non-structural protein NSs, a peculiarity is that, whilst N is produced from RNA transcribed from the genome, the message for NSs is transcribed from the antigenome. Superficially this is a little counter-intuitive; NSs is responsible for the rapid abrogation of the interferon system and thus would, presumably, be required as early as possible upon infection. Why force the virus to produce the antigenome before NSs can be produced?
A recent paper by Brennan, Welch and Elliott has addressed this by swapping around N and NSs, such that NSs is translated from the antigenome, and N from the RNA transcribed from the antigenome.

Generation of the swap virus: the (anti)genomic strand from which N and NSs are transcribed is 'swapped'.

In addition to the virus with wild-type (vaccine strain, MP12) and 'swap' virus, they also made viruses where NSs was substituted with EGFP. Now they had a panel of viruses including ones where NSs (or EGFP) were expressed first, before N. When they tested the growth of each virus, the swap viruses all grew poorly compared to the wild-type. This was regardless of whether the cells were either interferon competent (A549), incompetent (BHK21) mammalian cells, or indeed various insect cell lines.
Growth of 'swap' vs wt MP12 RVFV: In all cases, all swap viruses (filled circles) grow with lower efficiency compared to MP12 (A:mammalian cells; C. insect cells).

The lower apparent rate of replication was reflected in the amount of protein. As time progressed, the amount of protein accumulated by the swap virus was lower, although as expected the NSs protein was on this occasion produced before the N protein; opposite to what occurs with the wt virus. The accumulations of NSs were also much more substantial than with wt. However, although there was much more NSs (and less N) than wt, significant amounts of the glycoprotein (Gn) were not detected until 48 hours after infection, compared to 18-24 in the case of wt. In the case of the swap virus with EGFP in place of NSs, Gc was virtually undetectable even at 48h.
In wt RVFV, NSs assembles into filaments that are localised to the nucleus. Rather oddly, in the case of the swap virus, these were much thicker than the wt, with additional evidence of some NSs in the cytoplasm. These alterations in NSs behaviour appeared not to affect the functions of NSs in either inhibiting both host protein synthesis and host RNA synthesis. Given its 'shut-off' function, it is intriguing that increasing the abundance of NSs had no additional effect upon the intensity or rate at which protein shut-off occurs.  
Shut-off of host cell macromolecular synthesis: A: radioactive methionine/cysteine incorporation at different times post-infection; less label =  less protein = shut-off. B: shut-off of RNA synthesis (newly synthesised RNA is green, red is virus).

When they looked at the targets of NSs that result in shut-off, p62 was inhibitted by the swap virus (although more slowly), just as the wt but, bizarrely, PKR appeared to be largely unaffected by the swap virus, in contrast to the wt virus where PKR levels dropped from around 5 hours onwards. Overall, it would seem that, whilst it still does, the swap virus is a bit less efficient at the shut-off. As a result, it is a little surprising that the swap virus results in the induction of less IFN than the wt virus in A549 cells.

One thing the authors did tease apart is the relative number of genome:antigenome copies in both the cell and virion fractions. The swap virus was found to transcribe much more NSs RNA compared to the wt equivalent, suggesting that increased activity of the relative promoters is responsible for the dramatic amount of NSs observed with the swap virus. Such an excess of RNA may have overwhelmed the control by RNAi in insect cells, resulting in cytopathogenic effect in infected insect cells (in contrast to wt virus, which establishes a persistent infection). One interesting finding is that more antigenome than genome copies are packaged in swap virus virions. Does this reflect the abrogation a specific packaging process, or simply the abundance of genome:antigenome copies in the cytoplasm when packaging occurs? Considering the swap virus has N transcribed from the antigenome, the increase in antigenome packaging may actually overcome some of the temporal regulation achieved by swapping the N from the genomic to the antigenomic transcipt. It seems a bit more work  is required here. Virions have been shown to contain just 3 segments of RNA. If more of the virions have an antigenomic S segment, then the number of particles per infectious unit will necessarily be increased. It may be that such differences in the make-up of the viral population can explain some of the characteristics of the swap virus.

Of course such attenuating features - at least in vitro - may contribute towards the rational development of a vaccine. The most tempting feature of the swap virus described here is the fact that the virus cannot persist in insect cells and would thus be somewhat resistance to transmission; a key consideration in live arbovirus vaccines.

So, genome organisation is important. Ultimately this is not surprising: there's a reason it's like it is.

Brennan, B., Welch, S., & Elliott, R. (2014). The Consequences of Reconfiguring the Ambisense S Genome Segment of Rift Valley Fever Virus on Viral Replication in Mammalian and Mosquito Cells and for Genome Packaging PLoS Pathogens, 10 (2) DOI: 10.1371/journal.ppat.1003922

Sunday, 16 February 2014

Viral polymerases and pathogenesis
Read an introduction to viral evolution and you'll pretty quickly come across a sentence equating to 'viruses mutate and evolve fast and this is because their polymerases lack proofreading ability'. Sure, but that's a big generalisation. Large DNA viruses, for example, tend to have polymerases that are much less error-prone (again a generalisation). Nevertheless, in the world of RNA viruses it is a good rule of thumb that the polymerases tend to be error-prone when copying their genetic code. Superficially, such apparent laxity would appear to be detrimental to virus survival. However, if faithful replication of a nucleic acid was important, then the replication would indeed be faithful, but virus evolution occurs at a population level as opposed to the individual.

The viral sequences in GenBank are consensus sequences, in essence a read-out which represents the most commonly occurring base at each particular position in the genome. In a sense the concept of the consensus can be somewhat misleading: if RNA viruses have a mutation rate that is sufficiently great that it cannot copy a genome without making an error, then it's conceivable that the consensus per se does not, in reality, actually exist. Instead, a virus is a population of sequences evolving together, exploring sequence space with a great level of diversity.
Virologists tend to roll out the term quasispecies to describe this, but there remains some debate as to the validity of this theory, certainly in relation to Eigen's initial proposal.

Survival of the flattest: when mutation rates are low, all viruses accumulate together; if the peak is steep and narrow, then a low mutation error results in all of the viruses being exceptionally fit, (A outcompetes B). If mutation rates are high, then fitness is spread, in which case a spread of highly fit viruses is preferable to an extremely fit virus surrounded by viruses of low fitness (B outcompetes A, from Wilke 2005). 
Having such diversity means that the virus can tackle things that get in the way of progress, most obviously an immune response; if the response impacts upon the fitness of one sequence, then there's another that is capable of preserving the virus' existence. In order to generate such diversity, a virus must necessarily make mistakes and thus viruses tend to live quite close to the maximum permitted error rate for a particular lifestyle. Too much error, and the virus suffers error catastrophe and becomes extinct, as there are insufficient fit sequences. Too little error though, and sufficient diversity becomes hard to generate.

A paper came out recently describing a variant of Chikungunya virus (CHIKV) with altered levels of fidelity. A CHIKV mutant had previously been isolated by growing the virus in the presence of the antiviral ribavirin, which resulted in a virus with increased fidelity. This virus had a single mutation in the viral polymerase. The authors systematically changed this amino acid and looked at the effect it had. Of the 19 options attempted, 12 viruses were viable.

Figure 1 Mutagenizing position 483 variants allows isolation of mutator variants.
Mutator variants: A. the impact of different amino acids on the susceptibility to ribavirin treatment. B. mutation frequencies of selected variants compared to wild-type virus; G and W result in increased rates of mutation. C. average diversity of confirmed fidelity variants at each point across the genome; anti-mutator variant with a Y results in lower diversity across the genome. D. due to the greater diversity, mutator strains are better able to escape virus neutralisation by antibody (CHK-102).  
If these viruses have altered fidelity, then they should have different sensitivities to ribavirin, and indeed some viruses were less sensitive to ribavirin (antimutator strains) whereas some were more sensitive to ribavirin (putative mutator strains). When they checked for sequence diversity, they found a matching trend that sensitivity to ribavirin correlated with diversity, i.e. mutator viruses that make mistakes are susceptible to the treatment. A more highly mutated genome would in theory result in more non-viable/unfit genomes, and indeed they found that the progeny virus population of mutator strains was less infectious, even though their replication in mammalian cells was largely unaffected. When they injected some of the viruses into mice, there was a correlation between the frequency of mutation and viral loads. In line with the in vitro data, the mouse model revealed a decrease in pathogenicity associated with the increased error rates of mutator strains.

Whilst mutator strains replicated fine in mammalian cells, they replicated poorly in mosquito cells. As might be expected under such pressure, in vivo infections of mosquitoes, as well as passage in insect cells, resulted in a reversion of the mutator strains to a more wild-type level of fidelity. It would be interesting to see whether these viruses could be transmitted to subsequent hosts via mosquito bites.

Mosquito infections: Aedes  albopictus (A-C) or Aedes aegypti (D-F) mosquitoes were fed blood containing wild-type (WT) or mutator versions of CHIKV. Virus levels of all viruses was equivalent in the 

Another way in which they confirmed the impact of the changes associated with the polymerase fidelity and its impact upon virus phenotype was to specifically alter the equivalent position in another alphavirus: Sindbis virus (SINV). When they tested the mutated version of SINV, they found the same outcome, notably relatively little impact upon replication levels in mammalian cells, attenuation of pathogenicity in mouse models, lowered titres in insect cell cultures, and reversion to 'wild-type' in mosquito infections, thus confirming the importance of the mutator phenotype.

Mutator (and antimutator) viruses such as these may offer ways in which we can study further how viruses live close to the error threshold - perhaps with the prospect of adding evidence for or against the 'quasispecies' concept.
But replication competent virus but with attenuated phenotype? This rings the vaccine bell. At least potentially. There's more to do, and the authors highlight this, in particular with regard to how the viruses under selective pressure in vivo will behave and evolve in different hosts. But they're certainly an intriguing prospect.

Rozen-Gagnon, K., Stapleford, K.A., Mongelli, V., Blanc, H., Failloux, A-B., Saleh, M-C., Vignuzzi, M. (2014). Alphavirus Mutator Variants Present Host-Specific Defects and Attenuation in Mammalian and Insect Models PLOS Pathogens DOI: 10.1371/journal.ppat.1003877